Kluyveromyces phaffii killer toxin active against wine spoilage yeasts: purification and characterization.

Abstract

The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93% and 80% identity to a beta-1,3-glucanase of Saccharomyces cerevisiae and a beta-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a beta-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (beta-1,3 and beta-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt.